This program seeks to obtain a fundamental understanding of the physical and chemical mechanisms by which DNA repair glycosylases locate and catalytically repair damaged bases in a vast excess of undamaged base pairs in genomic DNA. The driving force for these efforts is the belief that this mechanistic knowledge can be used to rationally engineer the specificities of these enzymes for biotechnology and biomedical applications, and will also lead to the design of useful small molecule inhibitors for antiviral therapies and other applications. The specific aims of this proposal are to (i) Establish whether uracil DNA glycosylase (UDG) locates damaged sites in DNA by a sliding or 3D search strategy. Considerable debate exists in the DNA repair field concerning the role of processive enzyme translocation along the DNA in the site location mechanism. We will perform experiments to quantify the mean translocation distance along the DNA, the lifetime of specific and nonspecific protein-DNA complexes, the relative importance of short range and long range hopping between sites, and the efficiency of transfer between two damaged sites as a function of distance between sites, (ii) Discover how UDG traps extrahelical DNA bases that arise by spontaneous base pair breathing, and how this step leads to discrimination between normal and damaged base pairs in the genome. We will employ our recently developed imino proton exchange NMR methods to dissect the thermodynamic and kinetic features of normal and altered base pairs that promote or hinder extrahelical recognition by UDG. (iii) Understand the dynamic and structural differences between nonspecific and specific UDG-DNA complexes. We will use heteronuclear NMR methods to explore the structural and dynamic differences between free UDG and its complexes with undamaged and damaged DNA. These unique solution studies should provide the basis for how UDG exquisitely discriminates between undamaged and damaged DNA. (iv) Develop cell permeable inhibitors of human UDG (hUDG) based on high affinity inhibitors that promote base flipping or transition state interactions. Removal of uracil by hUDG is a key requirement in the life cycle of HIV-1. We will design cell permeable oligonucleotide inhibitors of hUDG using already established mechanistic principles of extrahelical base recognition and transition state mimicry.